Cells in most tissues are interconnected by cell-cell channels which allow the passage of electrolytes and small molecules from cell to cell. Aggregates of these channels are contained in the clustered intramembranous particles of gap junctions. The function of these channels has remained mysterious except in excitable cells where they are instrumental for impulse propagation. The objective of this research project is to understand the process of formation of cell-cell channels. This problem is addressed by functional expression of cell-cell channels in paired oocytes from cloned gap junction cDNA. With the oocyte assay conditions can be chosen where channel formation occurs at high rate (greater than 40 channel openings per second) from a pool of precursor. The oocyte assay will be used in combination with mutagenesis, synthetic peptides and the patch clamp technique to obtain information about molecular domains involved in the formation process. Channel formation at another level is proposed to be studied with gene transfer into mice (transgenics). These experiments are designed to yield information about the expression patterns of specific gap junction proteins during embryonic development. Furthermore, clues about the function of cell-cell channels in nonexcitable tissues can be expected from specific interference with gene expression.